Conservation genomics assessment of Tharp's bluestar (Amsonia tharpii) with comparisons to widespread (A. longilora) and narrowly endemic (A. fugatei) congeners

Abstract Land‐use change and habitat fragmentation are threats to biodiversity. The decrease in available habitat, increase in isolation, and mating within populations can lead to elevated inbreeding, lower genetic diversity, and poor fitness. Here we investigate the genetics of two rare and threatened plant species, Amsonia tharpii and A. fugatei, and we compare them to a widespread congener A. longiflora. We also report the first phylogenetic study of the genus Amsonia (Apocynaceae), including 10 of the 17 taxa and multiple sampling locations, to understand species relationships. We used a double digest restriction‐site associated DNA sequencing (ddRADseq) approach to investigate the genetic diversity and gene flow of each species and to create a maximum likelihood phylogeny. The ddRADseq data was mapped to a reference genome to separate out the chloroplast and nuclear markers for population genetic analysis. Our results show that genetic diversity and inbreeding were low across all three species. The chloroplast and nuclear dataset in A. tharpii were highly structured, whereas they showed no structure for A. fugatei, while A. longiflora lacked structure for nuclear data but not chloroplast. Phylogenetic results revealed that A. tharpii is distinct and sister to A. fugatei, and together they are distantly related to A. longiflora. Our results demonstrated that evolutionary history and contemporary ecological processes largely influences genetic diversity within Amsonia. Interestingly, we show that in A. tharpii there was significant structure despite being pollinated by large, bodied hawkmoths that are known to be able to carry pollen long distances, suggesting that other factors are contributing to the structure observed among A. tharpii populations. Conservation efforts should focus on protecting all of the A. tharpii populations, as they contain unique genetic diversity, and a protection plan for A. fugatei needs to be established due to its limited distribution.


| INTRODUC TI ON
Global change is a major cause of biodiversity decline and one of the many challenges to conservation (Haddad et al., 2015;Pereira et al., 2012;Sala et al., 2000).Pressures from land-use change and associated habitat loss, in particular, are often compounded and accompanied by fragmentation resulting in increased isolation and changes in the configurations of habitats and populations.
Fragmentation can contribute to the disruption of gene flow resulting in increased mating within populations (Haddad et al., 2015;Leimu-Brown et al., 2010;Young et al., 1996), limiting the exchange of novel diversity between populations, and a decrease in population size.In addition, effects from climate change such as increasing temperatures and drought will lead to higher mortality rates impacting population size, especially for plants distributed in arid regions (Hantson et al., 2021;McAuliffe & Hamerlynck, 2010;Parmesan & Yohe, 2003).As a result, genetic variation may change or decrease due to small population size, inbreeding, and genetic drift.This decrease can especially be an issue for endemic species which are often rare and threatened with extinction (Corlett & Westcott, 2013;Pecl et al., 2017).
Conservation genetics approaches assess the genetic status of populations by estimating genetic diversity, population connectivity, and effective population size, which offers insight into processes impacting population dynamics for these species and guides their management (Frankham et al., 2002;Lande, 1988;Soulé, 1985;Theissinger et al., 2023).Species and populations that are small and impacted by land-use change and fragmentation can have lower genetic diversity and increased rates of inbreeding (Aguilar et al., 2008(Aguilar et al., , 2019;;Cole, 2003;González et al., 2020;Honnay & Jacquemyn, 2007;Vellend, 2004), which can increase the extinction risk for threatened species through inbreeding depression, and loss of genetic diversity through drift (Brook et al., 2002;Frankham, 2005).However, gene flow (pollen or seed movement) can offset these impacts by increasing genetic variation and limiting inbreeding.Panmictic, genetically cohesive populations do not typically suffer from inbreeding depression and low genetic diversity (Charlesworth, 2003).
Populations that are connected through gene flow are potentially buffered from fragmentation (Ellstrand, 1992;Finger et al., 2014;Skogen et al., 2016).In plants, gene flow occurs through pollen and seed dispersal and the vectors of each may have distinct and different impacts on patterns of genetic diversity.Pollen transfer and seed dispersal can potentially rescue fragmented populations from low genetic diversity and inbreeding if at least one mode promotes gene flow with stable and intact populations (Frankham, 2015;Richards, 2000).Biotic pollination is known from nearly 90% of flowering plants (Ollerton et al., 2011) and the size and foraging behavior of pollinators can impact mating events and resulting patterns of genetic diversity, with larger animals carrying pollen longer distances (Gamba & Muchhala, 2020).For example, larger pollinators such as hawkmoths (Sphingidae) are capable of foraging longer distances which can create genetic cohesion among plant populations (Finger et al., 2014;Lewis et al., 2023;Skogen et al., 2019).By contrast, smaller pollinators such as bees, forage close to nesting sites and therefore over smaller areas, which can limit within and between population gene flow in the plants they visit (Dellinger et al., 2022;Greenleaf et al., 2007;Hasegawa et al., 2015).Bees also groom pollen from their bodies, limiting pollen carryover.Much like pollen movement, seed dispersal can promote connectivity (animal dispersal) or divergence (e.g. via gravity dispersal; Howe & Smallwood, 1982;Levin et al., 2003).
When the mode of dispersal differs between pollen and seed dispersal, the patterns of genetic diversity will likely differ between nuclear and plastid DNA, which can help identify the main driver of gene flow in the system.
Understanding how ecological, historical, and geographic variables contribute to genetic diversity of populations and species is important for those needing management.However, it is important to recognize that the genetic patterns of a species can often be driven by evolutionary constraints and historical factors, rather than contemporary process.For these reasons, comparisons of related species are encouraged to control for evolutionary history and identify species-specific differences (Gitzendanner & Soltis, 2000;Keller et al., 2013;Lanes et al., 2018;Ouborg et al., 2006;Soltis & Gitzendanner, 1999;Spence et al., 2021).Such approaches provide a phylogenetic perspective, which can also be critical for understanding species boundaries and can be used as a conservation framework.Closely related and recently diverged species share a common ancestor by descent and can therefore be genetically and morphologically similar.A phylogenetic approach can help to understand species relationships and species limits when genetic changes outpace morphological variation (Cohen & Schenk, 2022;Frankham, 2010;Hey et al., 2003;Purvis et al., 2005).In addition, a phylogenetic perspective can help to uncover hidden admixed or hybrid populations (Hibbins & Hahn, 2022).Furthermore, comparisons of congeners to threatened species help to determine the extent of extinction risk (Spielman et al., 2004).A phylogenetic perspective allows us to address species delimitation questions while also comparing meaningful population genetic parameters and both can be used in conservation management.
Here we compare patterns of genetic diversity, gene flow, and effective population size among three species in the plant genus Amsonia (Apocynaceae) to determine if rare taxa are genetically depauperate and experience limited gene flow.Two species, A. fugatei (Fugate's bluestar) and A. tharpii (Tharp's bluestar), are rare (known from three and seven localities, respectively) while A. longiflora (tubular bluestar) is more widespread than the other two species (Figure 1d).All three species have white, tubular flowers, with A. longiflora having longer floral tubes and larger corolla diameters than A. tharpii and A. fugatei, which have similar floral tube lengths and corolla diameters (Figure 1a-c (Figure 1e,f; Pierre et al., 2020), where all seven of the extant localities of A. tharpii occur (demographic monitoring by the Bureau of Land Management of the five localities included in this study started in 2017; Yannayon et al., 2021).As a result of increased pressures from oil and gas development, drought, and demographic decline, A. tharpii is a candidate for listing under the US Endangered Species Act.The ranges of A. fugatei and A. longiflora largely do not occur in the Permian Basin.Amsonia fugatei is only know from three localities and A. longiflora is considered secure.In addition to land-use change, climate projections suggest that this region will experience hotter and drier conditions with increased periods of intense drought (Archer & Predick, 2008;Petrie et al., 2014).Concerns about genetic diversity in A. tharpii stem from demographic declines since 2017, an increase in land-use change between localities, and climate change (Yannayon et al., 2021).In addition, because A. tharpii and A. fugatei are morphologically similar (floral tubes of similar length, similar corolla diameter; Figure 1a,b) despite being geographically isolated by the Sacramento and San Andres Mountains, we used a phylogenetic framework to elucidate species boundaries between these and other members of Amsonia.We hypothesize that due to the anthropogenic changes resulting in increased fragmentation in the intervening land between A. tharpii localities, they will have lower genetic diversity and higher inbreeding than the two secure Amsonia species.However, as all three species have floral traits consistent with hawkmoth pollination (white, tubular corollas), we predict that localities for each species will be genetically cohesive due to high gene flow.Indeed, other studies have shown that hawkmoths can travel long distances (Stockhouse, 1973) and population genetic studies of hawkmoth pollinated species show high rates of gene flow and little-to-no population differentiation (Lewis et al., 2023;Skogen et al., 2019).In addition, we hypothesize that A. tharpii is genetically distinct from A. fugatei due to geographic isolation and that A. longiflora is more distantly related (McLaughlin, 1982).
Amsonia species are long-lived, perennial sub-shrubs with linear to ovate leaves, tubular white to blue corollas, and dry dehiscent fruits.Plants emerge from underground stems in early spring and typically flower in spring, and fruit in early summer.The three species have floral traits that are consistent with hawkmoth pollination: white flowers, long corolla tubes, scent (Fenster et al., 2004) and hawkmoths have been observed visiting both A. tharpii and A. longiflora (data not reported).Although the self-compatibility system is not known for the genus, self-incompatible systems are known from other taxa across the Apocynaceae (Coombs et al., 2009;Ollerton et al., 2019).These Amsonia species occur in and around the northern extent of the Chihuahuan Desert in New Mexico and west Texas on well-drained carbonate soils such as sand, limestone, and dolomite.Amsonia tharpii occurs across a highly fragmented landscape in southeastern New Mexico and west Texas (Figure 1a,d-f).Amsonia fugatei is a narrow endemic species distributed across relatively undisturbed habitat in central Socorro County, New Mexico (Figure 1b,d, McLaughlin, 1985) and is geographically separated from A. tharpii by the Sacramento and San Andres Mountains.Amsonia longiflora has the broad-
TA B L E 1 Locality information, individuals sampled (N S ), estimated census size (N C ), effective population size (N e ), and inbreeding coefficient (F IS ) for each locality.
groupings of individuals that vary in size and isolation.Samples were collected from across the known geographic extent for each of the five targeted localities.

| Sample collection
Leaf tissue was collected in the field and dried on silica gel from of all three species during 2020 and from two A. longiflora localities in 2022 (BLA and OLI).We sampled 10-30 individuals per location from five localities of A. tharpii, all three localities of A. fugatei, and six localities of A. longiflora (Figure 1d, Table 1).To control for geographic distance, we sampled A. longiflora from the western edge of its range, across a geographic extent similar to the distribution of the sampled A. tharpii localities.We sampled from locations that occurred near one another (10-45 km) and others more distant (45-200 km) across all species except A. fugatei due it being limited to three localities of which the farthest were separated by 45 km (Figure 1d).Our phylogenetic assessment included eight western and two eastern North American Amsonia species sampled from naturally occurring localities, herbarium vouchers, and living collections at botanic gardens (Table S1).

| DNA extractions/library prep
DNA extractions and two ddRADseq libraries were prepared at the Harris Genetics Laboratory at Chicago Botanic Garden.A modified CTAB protocol was used for DNA extraction (Doyle & Doyle, 1987) and all samples were visualized on electrophoresis gels and quantified with a Qubit v. were multiplexed with 48 individuals (or less).After multiplexing, each library was size selected using magnetic beads or with a Pippin Prep, targeting between 300 and 650 base pairs in length.Libraries were then amplified in 20 μL PCR reactions using a thermocycler and 15-20 cycles.Libraries were cleaned with magnetic beads and quantified before sequencing at the NUSeq Core facility at Northwestern University using the Illumina NovaSeq 6000 sequencer and with 150 bp (basepairs) paired end reads.

| Population genetics
All samples were demultiplexed, processed for mismatched sequences, cleaned, and trimmed to 100 bp with the process_radtags function in STACKS v. 2.55 (Rochette et al., 2019).Raw reads were trimmed to account for low quality and erroneous base pairs found at the end of the raw sequences.Parameters for calling SNPs were optimized using the "r80 rule" and by testing different values of n and M, while keeping m = 3 constant (Table S2, Paris et al., 2017).For optimization of each dataset, we first treated all the data as a single population to test different n and M values.The best parameter set was then chosen according to the most polymorphic loci and SNPs (Paris et al., 2017).We hypothesized that each location would be connected via gene flow and did not alter the -p value (number of populations) because this could have biased our results and called more unique loci/SNPs per population.To compare the threatened and non-threatened species while accounting for geographic distance and evolutionary history, two different datasets were used.
The first dataset, called "Individual Species SNPs", we obtained SNPs for each taxon separately.This dataset controlled for evolutionary differences among the three species to look for population-level divergences.The "Individual Species SNPs" dataset was separated by genome type to explore pollen and seed (nuclear genome) to only seed movement (plastid genome).To recover the plastid data from nuclear datasets we mapped demultiplexed reads to the Amsonia tabernaemontana reference chloroplast genome (Wang et al., 2023) using default parameters in BWA v. 0.7.12 and in SAMTOOLS v. 1.6 (Li et al., 2009;Li & Durbin, 2009).Those reads that did not map to the reference plastid genome were used for the nuclear dataset.The unmapped reads were treated as from the nuclear genome and processed in STACKS using the denovo_map.plpipeline, whereas the mapped reads were assumed to be from the chloroplast and SNPs were called using the ref_map.plpipeline applying the "r80 rule" and default settings.
The second dataset, "Shared SNPs", was used to test how genetic diversity (and inbreeding) relates to evolutionary history.Little is known about the evolutionary history of Amsonia, but closely related and recently diverged species share many morphological and genetic attributes (McLaughlin, 1982;Ngô & Applequist, 2023).
Here all three species were filtered in STACKS together, and therefore only SNPs shared across all three species were used to investigate genetic diversity and inbreeding.For all datasets, the best set of parameters was selected according to the highest number of polymorphic loci and total number of SNPs (Table S2).
VCFtools (Danecek et al., 2011) was used to filter each dataset for sequencing depth, sequence quality, minor allele frequency, and missingness before population genetic analyses.All nuclear datasets were filtered with a 90% missingness threshold allowing for only 10% missing data, a minor allele count of three, a min-mean DP of five and a max-mean DP of 50, a minDP of five and maxDP of 50, and maximum number of alleles of two.The plastid "Individual Species SNPs", datasets were only filtered with a 90% missingness threshold.Pairwise F ST was estimated using the Weir and Cockerham (1984) method for each locality pair across each species also using hierfstat (Goudet, 2005).We tested for correlations between population pairwise F ST and geographic distance (IBD) using dartR and Mantel test for significance.ADMIXTURE v 1.30 was used to infer population structure (pollen movement) across each species and was run with 20 replicates testing K values (genetic clusters) of 1-10 for A. tharpii and A. longiflora, and 1-6 for A. fugatei.A 15-fold cross-validation (CV) was performed for each replicate, and the most appropriate K value was selected according to the lowest averaged CV value.
ADMIXTURE results were visualized using the R package StructRly v 0.1.0(Criscuolo & Angelini, 2020).Lastly, we used Poppr v. 2.9.4 and calculated genetic dissimilarity matrices using default settings and the bitwise.dist()function and then converted and plotted these as minimum spanning networks with poppr.msn()and plot_poppr.msn()functions (Kamvar et al., 2014(Kamvar et al., , 2015) ) to explore plastid diversity (seed movement) within each species.

| Phylogeny
We addressed evolutionary relationships by estimating phylogenetic relationships using 62 Amsonia samples from 10 of the 17 currently recognized species, and from multiple locations whenever possible, to test for species monophyly (Table S1).The Python program ipyrad v 0.9.87 (Eaton & Overcast, 2020) was used to demultiplex, filter, and cluster reads, and to align ddRAD loci for phylogenetic analysis.
We clustered reads with an 85% similarity threshold and explored the effects of missing data by varying the number of samples required to have shared loci for it to be kept in the final alignment (Table S3).IQ-Tree was used to infer a model of DNA sequence evolution and a maximum likelihood (ML) phylogeny based on the concatenated DNA sequence matrix (Nguyen et al., 2015).Model finder was used within IQ-Tree to infer the most likely model of evolution based on AICc (Kalyaanamoorthy et al., 2017).The best fit model was then used to infer a ML tree, and node support was assessed with 100 nonparametric bootstraps (Nguyen et al., 2015).Two eastern Amsonia species were used as outgroups (Table S1).

| Genetic diversity, inbreeding, and effective population size
Optimization of STACKS parameters revealed that m = 3, M = 2, and n = 2 retrieved the most polymorphic loci and variant sites (SNPS) for the A. tharpii "Individual Species SNPs" dataset while m = 3, M = 2, and n = 3 had the most polymorphic loci and SNPs for the A. fugatei and A. longiflora datasets, and m = 3, M = 2, and n = 3 for the "Shared SNPs" dataset (Table S2).After filtering with VCFtools the "Individual Species SNPs" datasets recovered 1605 nuclear and 58 plastid SNPs for A. tharpii, 643 nuclear and 32 plastid SNPs for A. fugatei, and 956 nuclear and 57 plastid SNPs for A. longiflora.The "Shared SNPs" dataset retained 850 nuclear SNPs.Genetic diversity (expected heterozygosity) was low across both the "Individual Species SNPs" and "Shared SNPs" datasets (Figure 2).Amsonia fugatei and A. tharpii had a slightly higher genetic diversity than A. longifora in the "Shared SNPs" dataset and only A. fugatei had a higher genetic diversity than the other species in the "Individual Species SNPs" comparison (Figure 2a,b).Estimates of inbreeding were low across all species and populations (here after populations refer to genetic populations), ranging from 0.05 (A.fugatei) to 0.11 (A.tharpii) and 0.12 (A.longiflora) for the "Shared SNPs" datasets.Inbreeding coefficients were lower within the "Individual Species SNPs" data sets (0.06, A. tharpii -0.09, A. longiflora), and across A. tharpii populations (0.01 for BEN -0.09 for RED) and A. longiflora populations (0.04 for DEL -0.09 for BLA, Table 1).Overall, effective population size was low across most populations investigated (Table 1).Amsonia tharpii populations had the lowest N e and ranged from 6.5 (BEN) to 72.8 (RED).There was greater variation across A. longiflora populations with 29.5 (WOO) and 118.3 (ENG).Amsonia fugatei populations also had much higher N e values than any of the other Amsonia species, ranging from 84.8 (FIF) to 217.2 (SEV, Table 1).

| Population structure
There was no evidence of isolation by distance in any species (A.tharpii: r = 0.31, p = 0.34; A. fugatei r = 0.71, p = 0.33, A. longiflora r = 0.71, p = 0.07; Figure S1), however of all three species examined, A. longiflora had overall the lowest pairwise distance for any given distance (Table S4).Within A. longiflora populations the pairwise distance ranged from 0.04 (DEL and AZO, 61 km) to 0.13 (WOO and ENG,197 km), and over a similar geographic extent, the pairwise distance of A. tharpii ranged from 0.08 (RED and CAP, 32 km) to 0.18 (BEN and TEX,254 km).Last, in A. fugatei populations the pairwise distance (10-45 km) showed similar genetic distance, ranging from 0.08 to 0.10 (Table S4).
The ADMIXTURE results suggested that K = 5 was the most likely number of genetic clusters for A. tharpii (Figure 3, Figure S2).In comparison, A. longiflora and A. fugatei cross-validation results suggested K = 2 as the most likely number of genetic clusters (Figure 3, Figure S2).Within A. longiflora there was genetic structuring between western (ENG and OLI) and the eastern New Mexico populations (AZO, BLA, DEL, and WOO).The minimum spanning networks used to assess seed dispersal suggested that populations of A. tharpii and A. longiflora were geographically structured, however the A. fugatei network lacked structure (Figure 4).In addition, a few individuals from the nearest A. tharpii populations at RED and CAP contained similar plastid diversity that may represent an ancestral haplotype, and the TEX population appeared to be derived from the RED population (Figure 4a).

| Phylogeny
All three alignments retained similar topologies, however, the alignment with the most loci (50819) and missing data (61.5%) was also the best supported with bootstrap values (Table S3).The Amsonia backbone topology and most species relationships were strongly supported, with the only exception being A. palmeri (Figure 5).Two samples of A. palmeri, one from NM and the other from TX, were recovered as serially sister to the federally listed and narrow endemic A. kearneyana from southern Arizona (Figure 5).Amsonia tharpii was recovered as monophyletic and sister to A. fugatei (Figure 5).
Together these species were sister to a clade that included A. tomentosa from the Mojave Desert and A. arenaria from the Chihuahuan Desert.Amsonia longiflora was recovered as sister to the morphologically similar A. grandiflora and together they were sister to A. palmeri and A. kearneyana, but distantly related to A. tharpii and A. fugatei (Figure 5).The populations of A. tharpii, A. fugatei, and A. longiflora were all recovered as monophyletic but some with lower support values (Figure 5).

| DISCUSS ION
Using a comparative approach, we showed that genetic parameters within populations were equivalent across the three species of Amsonia with similar floral morphology and seed traits.Genetic diversity was not lower, and inbreeding was not higher for the threatened species, A. tharpii or A. fugatei, when compared to the secure congener, A. longiflora (Figure 2, Table 1), however the effective population sizes for all the populations were below recommended conservation levels for all species (Table 1, Frankham et al., 2014).
These results allow us to reject our hypotheses that genetic diversity is lower in the threatened A. tharpii.By contrast we found that there were differences in between population parameters, with A. fugatei F I G U R E 2 Genetic diversity (expected heterozygosity) for (a) "Shared SNPs" and (b) "Individual Species SNPs" datasets and across (c) Amsonia tharpii and (d) A. longiflora populations."Individual Species SNPs" not shown for A. fugatei.Populations are plotted as they occur from west to east.
and A. longiflora appearing to be genetically cohesive for nuclear data, likely driven by pollen movement, while A. tharpii showed strong structure with both nuclear and plastid genomes (Figures 3   and 4).Finally, our phylogenetic dataset supported our final hypothesis by revealing A. tharpii and A. fugatei to be genetically distinct as was the case for other members of the genus from western North America (Figure 5).
The similar levels of genetic diversity recovered across all populations and species, regardless of rarity, supports previous studies (Gitzendanner & Soltis, 2000) which suggest evolutionary history plays a large role in controlling genetic variation across closely related species (Figure 5).In comparison, a population genetic study on the eastern North American A. ludovicana, recovered higher genetic variation (H O = 0.36-0.54)and similar inbreeding coefficients (F IS = 0.01-0.13)(Figure 2, Table 1, Smallwood et al., 2018).The differences in genetic diversity could be explained by data collection method (ddRADseq vs. microsatellites) (Hodel et al., 2017;Sunde et al., 2020), sampling collection bias (Hale et al., 2012;Rosenberger et al., 2021), population sizes, or a result of being more distantly related to the western Amsonia species.By contrast, the inbreeding coefficients across populations of all four taxa (A.ludovicana and A. tharpii, A. fugatei, and A. longiflora) were low and consistent with a self-incompatible breeding system, which is known in the Apocynaceae (Gibbs, 2014;Lipow & Wyatt, 1999;Shuttleworth & Johnson, 2006).Despite relatively low effective population size, similar estimates of inbreeding and genetic diversity across taxa suggest these represent historically stable population sizes.While The one major difference we did identify is in connectivity between populations by species, with the lowest genetic distances between populations of A. longiflora with the nuclear data, and for between populations of A. fugatei for plastid data (Table S4, Figure 4b).Because all three taxa share floral traits and hawkmoths have been recorded visiting A. tharpii and A. longiflora, (data not shown), we hypothesized that populations would be genetically similar and lack population structure across the nuclear dataset and genetically distinct (structured) in the plastid dataset.
Instead, we found that A. tharpii was structured for both datasets (Figures 3c and 4a, Table S4).Despite the relative proximity of some A. tharpii populations (CAP and RED (32 km), BEN and CPC (47 km), Figure 1d, Table S4), pollen and seed movement appear to be limited to within populations.This contrasts with what other studies of hawkmoth-pollinated taxa have found (Cisternas-Fuentes et al., 2022;Finger et al., 2014;Rhodes et al., 2017;Skogen et al., 2019).For example, a study from the same region (Lewis et al., 2023) documented gene flow among populations of the hawkmoth pollinated Oenothera hartwegii subsp.filifolia separated by 13-400 km.This pattern is partially explained by the presence of intervening populations between the sampled populations facilitating gene flow via a steppingstone pattern.Only two known, unsampled populations occur in proximity to BEN and CPC, but even with potential steppingstone populations A. tharpii appears to be highly structured.Our results are consistent with other studies of plants that occupy small patches and have edaphic or unique ecological habitats whereby low genetic diversity and population structure have been documented (Barbará et al., 2008;Loveless & Hamrick, 1984;Matesanz et al., 2018;Moore et al., 2014;Ramirez-Barahona et al., 2014) even when visited by large nectar-feeding birds pollinators assumed to travel long distances (Bezemer et al., 2016;Nistelberger et al., 2015).While our results indicate that A. tharpii is experiencing genetic drift and divergence may be One interesting outcome was that the plastid haplotype networks revealed limited seed dispersal for A. tharpii and A. longifora, but not for A. fugatei.Shorter seed dispersal distances are expected to increase population structure, and all Amsonia species have large, seeds that are primarily gravity dispersed (Gelmi-Candusso et al., 2017;Loveless & Hamrick, 1984;Schaal et al., 1998), although their "corky" coating might suggest they have the capacity to float during flooding events.Notably, within A. tharpii, individuals from CAP and RED shared similar plastid diversity, and TEX appeared to be derived from the RED population, suggesting long-distance seed movement or the existence of intervening populations at some point in the past (Figure 4a).In contrast, all three populations of A. fugatei Considering population genetic parameters in light of evolutionary history revealed similar genetic diversity and inbreeding between closely related sister species, A. tharpii and A. fugatei, and the distantly related A. longiflora (Figures 2 and 5).This is not surprising as comparisons of population genetic parameters between widespread and narrow endemic congeners are often correlated because of a shared derived ancestry (Gitzendanner & Soltis, 2000) and while contemporary processes impact genetic diversity measures, our results suggest this has yet to have occurred for the A. tharpii populations.We hypothesized that A. tharpii would be genetically distinct from A. fugatei based on geographic isolation, and our results support this (Figure 5).Amsonia tharpii and A. fugatei are separated by 250 km of distance in addition to both the Sacramento and San Andres Mountains which appeared to be the only barrier to pollen movement within A. longiflora populations (Figure 4).Amsonia palmeri was the only species recovered as non-monophyletic (Figure 5).Amsonia palmeri has a wide ).These three species occur in or near the northern extent of the Chihuahuan Desert, where much of this landscape has been impacted by land-use change.The most dramatic change in the last 10-15 years has been a result of the extensive oil and gas development that has occurred in the Permian Basin F I G U R E 1 Inflorescences of (a) Amsonia tharpii, (b) A. fugatei, (c) A. longiflora.(d) Map of sampling localities for population genetics study.We sampled five localities from A. tharpii, six from A. longiflora and all three A. fugatei localities.Aerial photos of land-use change between (e) 1985 and (f) 2024 near the Red Lake locality.
est distribution extending from central New Mexico to central Texas (Figure 1c,d).Additionally, two of the A. longifora localities (ENG and OLI) occur west of the Sacramento Mountains and the remaining localities used in this study to the east and south of these mountains.Localities of A. tharpi are comprised of smaller 2.0 prior to library prep.Sequencing library preparation followed Peterson et al. (2012) with modifications from Diaz-Martin et al. (2023) and others noted below.We normalized each DNA sample to 20 ng/μL prior to digestion.Normalized genomic DNA was then digested for 24 h with two restriction enzymes, EcoRI and MspI.Following digestion, P1 adapters were ligated to DNA fragments associated with the EcoRI enzyme.Libraries Genetic diversity (expected heterozygosity) and the inbreeding coefficient (F IS ) were calculated in RSTUDIO v. 4.1.2using hierfstat v. 0.5-11 and dartR v. 2.0.4(Goudet, 2005;Gruber et al., 2017; R   Core Team, 2021).NeEstimator v. 2 was used to calculate effective population size (N e )(Do et al., 2013).Default settings were used for NeEstimator along with a Linkage Disequilibrium model that assumed random mating events and a critical value of 0.05.Patterns of gene flow were explored from the nuclear derived datasets with pairwise F ST , isolation by distance (IBD), and with the maximum likelihood-based program ADMIXTURE(Alexander et al., 2009).
our results suggest A. tharpii is not suffering from genetic erosion, individuals can be long-lived, making it difficult to determine if the patterns revealed in our dataset represent a more contemporary or a historic legacy of gene flow.Comparisons of genetic diversity and gene flow between parent and offspring generations would better determine the impact of more recent land-use change and resulting habitat fragmentation and extreme drought events.
due to habitat fragmentation, comparisons of historic and contemporary gene flow (parent-offspring comparisons) are needed to assess the extent to which this is likely.While the landscape surrounding sampled populations has been altered by oil and gas development in recent years, it is difficult to distinguish between F I G U R E 3 ADMIXTURE barplots for (a) Amsonia fugatei (K = 2), (b) A. longiflora (K = 2), (c) A. tharpii (K = 5).Populations of A. tharpii and A. longiflora are plotted from west to east as they are distributed.its affects and those of major drought events potentially due to climate change in the region that have occurred over the same timeframe(Archer & Predick, 2008;Briggs et al., 2020;Petrie et al., 2014).The lack of adequate precipitation from annual summer monsoon events may lead to changes in population size due to mortality and delayed emergence, reductions in number of flowers, and changes in co-flowering species diversity, which provide supplemental resources for pollinators (both floral resources for adults and vegetative for larval stages of some taxa).Over time these factors may impact population genetic parameters.Last, we cannot rule out demographic patterns, life history, and density dependent foraging patterns as a factor contributing to population structure.
shared haplotypes.Amsonia fugatei occurs along the Rio Grande River in central New Mexico and the lack of structure may represent a single derived haplotype or recent migration (>15,000 years ago) following extreme flooding events(Repasch et al., 2017) as seeds may be dispersed via water as seeds float.
distribution from northwestern AZ to southwestern NM, thru the Mexican states of Chihuahua and Sonora, and into eastern TX (McLaughlin, 1982; Ngô & Applequist, 2023) and additional F I G U R E 4 Minimum spanning networks showing plastid diversity for (a) Amsonia tharpii, (b) A. fugatei, and (c) A. longiflora.Numbers within circles indicate how many samples possess identical chloroplast type.Line color and width represent genetic distance between individuals and populations.Scale bar represents genetic distance.